Hepatic fibrogenesis is mediated by the activation of hepatic stellate cells (HSC), a process that is characterized by a dramatic phenotypic transformation, wherein HSC acquire myofibroblastic features and an enhanced capacity to proliferate. Growth factors such as platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) play an important role in HSC activation. Cell proliferation is ultimately governed at the level of the cell cycle, and is dependent on the balance of positive (cyclins, cyclin-dependent kinases; CDK) and negative (p27Kip1) regulatory cell-cycle proteins. Several intracellular signaling pathways have been implicated in transducing mitogenic signals from growth factor receptors to the cell-cycle machinery, including the extracellular signal-regulated kinase (ERK) pathway, the phosphatidylinositol-3-kinase (PI3K) pathway and the p70 S6 kinase (PSK) pathway. However, little is known about the potential roles of the ERK, PI3K, and PSK signaling pathways in regulating cell-cycle proteins and the activation process in HSC. Therefore, the long-term goal of this project is to gain a better understanding of the expression of regulatory cell-cycle proteins and the intracellular signaling pathways that drive proliferation and activation of HSC. Accordingly, in Specific Aim 1, we will test the hypothesis that PDGF and IGF-1 stimulate HSC proliferation by increasing cyclin levels and CDK activity, or by inducing the degradation of the CDK inhibitor p27Kipl. We will also assess the role of the ERK pathway in mediating these effects, and in HSC activation. In Specific Aim 2, we will determine whether PDGF and IGF-1 stimulate PI3K and if this pathway contributes to their mitogenic effects (including changes in cell-cycle proteins)and to the activation of HSC (including collagen expression). In Specific Aim 3, we will test the hypothesis that PSK plays a role in the effects of PDGF and IGF-1 on cell-cycle proteins in HSC, and we will test the effect of the selective pathway inhibitor, rapamycin, on HSC activation in vitro. To extend the findings in Specific Aim 3 to the in vivo setting, in Specific Aim 4, we will use well-characterized hepatic injury models to test the hypotheses that rapamycin has suppressive effects on HSC activation and fibrogenesis, and can reverse these processes even when they are fully established. These proposed experiments will substantially increase our knowledge of the intracellular signaling pathways that drive HSC proliferation and activation. Since activated HSC play a key role in hepatic fibrogenesis, these experiments will contribute to an improved understanding of the basic biochemical processes underlying hepatic fibrosis.